Exploring Structure-Activity Relationships of Niclosamide-Based Colistin Potentiators in Colistin-Resistant Gram-Negative Bacteria.

Colistin is primarily used as a last resort antibiotic against highly resistant Gram-negative bacteria (GNB). Rising rates of colistin resistance, however, may limit future use of this agent. The anthelmintic drug niclosamide has been shown to enhance colistin activity in combination therapy, but a detailed structure-activity relationship (SAR) for niclosamide against GNB has yet to be studied. A series of niclosamide analogs were synthesized to perform an SAR, leading to the discovery of a lead compound that displayed comparable colistin-potentiating activity to niclosamide with reduced cytotoxicity. Overall, this work provides important insights into synthetic strategies for the future development of new niclosamide derivatives and demonstrates that toxicity to mammalian cells can be reduced while maintaining colistin potentiation.

Dioctanoyl Ultrashort Tetrabasic ß-Peptides Sensitize Multidrug-Resistant Gram-Negative Bacteria to Novobiocin and Rifampicin.

Recently reported peptidomimetics with increased resistance to trypsin were shown to sensitize priority multidrug-resistant (MDR) Gram-negative bacteria to novobiocin and rifampicin. To further optimize proteolytic stability, ß-amino acid-containing derivatives of these compounds were prepared, resulting in three dioctanoyl ultrashort tetrabasic ß-peptides (dUSTBßPs). The nonhemolytic dUSTBßP 3, comprised of three ß3-homoarginine residues and two fatty acyl tails eight carbons long, enhanced the antibacterial activity of various antibiotics from different classes. Notably, compound 3 retained the ability to potentiate novobiocin and rifampicin in wild-type Gram-negative bacteria against MDR clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. dUSTBßP 3 reduced the minimum inhibitory concentration of novobiocin and rifampicin below their interpretative susceptibility breakpoints. Furthermore, compound 3 exhibited improved in vitro stability (86.8 ± 3.7% remaining) relative to its α-amino acid-based counterpart (39.5 ± 7.4% remaining) after a 2 h incubation in human plasma.

Dilipid Ultrashort Tetrabasic Peptidomimetics Potentiate Novobiocin and Rifampicin Against Multidrug-Resistant Gram-Negative Bacteria.

The development of new antibacterial agents and therapeutic approaches is of high importance to address the global problem of antibiotic resistance. Although antimicrobial peptides are known to synergize with certain antibiotics, their clinical application is limited by their systemic toxicity, protease instability, and high production cost. To overcome these problems, nine dilipid ultrashort tetrabasic peptidomimetics (dUSTBPs) were prepared consisting of three basic amino acids separated by a molecular scaffold, bis(3-aminopropyl)glycine, and were ligated to two fatty acids. Several nonhemolytic dUSTBPs were shown to enhance the activity of several antibiotics against pathogenic Gram-negative bacteria. More importantly, dUSTBP 8, consisting of three l-arginine units and a dilipid of 8 carbons long, potentiated novobiocin and rifampicin consistently against multidrug-resistant (MDR) clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae. Preliminary studies suggested that dUSTBPs were likely to potentiate antibiotics through outer membrane permeabilization and/or disruption of active efflux and that dUSTBP 8 exhibited enhanced resistance to trypsin in comparison to the previously described di-C9-KKKK-NH2 antibiotic potentiator. The antibacterial activity of rifampicin and novobiocin was enhanced by dUSTBP 8 comparable to other known outer membrane permeabilizing potentiators including the gold standard polymyxin B nonapeptide. Our results indicate that ultrashort tetrabasic peptidomimetics are potent adjuvants that repurpose novobiocin and rifampicin as potent agents against priority MDR Gram-negative pathogens.

Repurposed Antimicrobial Combination Therapy: Tobramycin-Ciprofloxacin Hybrid Augments Activity of the Anticancer Drug Mitomycin C Against Multidrug-Resistant Gram-Negative Bacteria.

The lack of therapeutic options to treat infections caused by multidrug-resistant (MDR) pathogens, especially Gram-negative bacteria, is apparent. Therefore, it is imperative to develop new strategies to address the problem of antimicrobial resistance. Repurposing non-antibiotic commercial drugs for antimicrobial therapy presents a viable option. We screened six anticancer drugs for their potential use in antimicrobial therapy. Here, we provide in vitro evidence that suggests feasibility to repurpose the anticancer drug mitomycin C against MDR Gram-negative bacteria. We also demonstrated that mitomycin C, etoposide and doxorubicin were affected by drug efflux in Pseudomonas aeruginosa. In combination with a tobramycin-ciprofloxacin antibiotic hybrid (TOB-CIP), the antibacterial activity of mitomycin C was enhanced against MDR clinical isolates of P. aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. In fact, 4 µg/mL (3 µM) TOB-CIP reduced the minimum inhibitory concentration of mitomycin C to ≤1 µg/mL against MDR Gram-negative bacteria, except A. baumannii. We showed that synergy was inherent to TOB-CIP and that neither tobramycin nor ciprofloxacin individually synergized with mitomycin C. Our finding supports identifying adjuvant partners for mitomycin C, such as TOB-CIP, to enhance suitability for antimicrobial therapy.

Amphiphilic nebramine-based hybrids Rescue legacy antibiotics from intrinsic resistance in multidrug-resistant Gram-negative bacilli.

The inability to discover novel class of antibacterial agents, especially against Gram-negative bacteria (GNB), compel us to consider a broader non-conventional approach to treat infections caused by multidrug-resistant (MDR) bacteria. One such approach is the use of adjuvants capable of revitalizing the activity of current existing antibiotics from resistant pathogens. Recently, our group reported a series of tobramycin (TOB)-based hybrid adjuvants that were able to potentiate multiple classes of legacy antibiotics against various MDR GNB. Herein, we report the modification of TOB-based hybrid adjuvants by replacing TOB domain by the pseudo-disaccharide nebramine (NEB) through selective cleavage of the α-d-glucopyranosyl linkage of TOB. Potent synergism was found for combinations of NEB-based hybrid adjuvants with multiple classes of legacy antibiotics including fluoroquinolones (moxifloxacin and ciprofloxacin), tetracyclines (minocycline), or rifamycin (rifampicin) against both wild-type and MDR P. aeruginosa clinical isolates. We also demonstrated that a combination of the optimized NEB-CIP hybrid 1b and rifampicin protects Galleria mellonella larvae from the lethal effects of extensively drug-resistant (XDR) P. aeruginosa. Mechanistic evaluation of NEB-based hybrid adjuvants revealed that the hybrids affect the outer- and inner membranes of wild-type P. aeruginosa PAO1. This study describes an approach to optimize aminoglycoside-based hybrids to yield lead adjuvant candidates that are able to resuscitate the activity of partner antibiotics against MDR GNB.

Polybasic peptide-levofloxacin conjugates potentiate fluoroquinolones and other classes of antibiotics against multidrug-resistant Gram-negative bacteria.

To address the rising threat of multidrug-resistant (MDR) bacteria, new therapeutic strategies must be developed. Efficacious drug combinations consisting of existing antibiotics and enhancer biomolecules called adjuvants offers a viable strategy. We have previously reported antibiotic hybrids consisting of tobramycin appended to different fluoroquinolones that possess potential as stand-alone antimicrobials as well as adjuvants. Herein, we report the synthesis of polybasic peptide-levofloxacin conjugates based on these tobramycin-fluoroquinolone hybrids. It was found that conjugating polybasic peptides to the fluoroquinolone levofloxacin, along with the addition of an aliphatic hydrocarbon tether, resulted in the ability of these compounds to potentiate fluoroquinolones and other antibiotics against MDR Gram-negative bacteria. The conjugates were able to potentiate ciprofloxacin, levofloxacin and moxifloxacin against MDR clinical isolates of Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and to a lesser extent, Acinetobacter baumannii. Preliminary data revealed that the conjugates interfered with active efflux of fluoroquinolones in P. aeruginosa. In addition, synergy was observed with a wide array of other antibiotics against P. aeruginosa, including those that suffered from restricted outer membrane penetration, suggesting that in addition to blocking active efflux, the polybasic peptide-levofloxacin conjugates possessed the ability to disrupt and permeabilize the outer membrane of Gram-negative bacteria.

Synergistic combinations of anthelmintic salicylanilides oxyclozanide, rafoxanide, and closantel with colistin eradicates multidrug-resistant colistin-resistant Gram-negative bacilli.

Repurposing nonantibiotic drugs for antimicrobial therapy presents a viable approach to drug discovery. Development of therapeutic strategies that overcome existing resistance mechanisms is important especially against those bacterial infections in which treatment options are limited, such as against multidrug-resistant Gram-negative bacilli. Herein, we provide in vitro data that suggest the addition of anthelmintic salicylanilides, including oxyclozanide, rafoxanide, and closantel, in colistin therapy to treat multidrug-resistant colistin-susceptible but more importantly colistin-resistant Gram-negative bacilli. As a stand-alone agent, the three salicylanilides suffered from limited outer membrane permeation in Pseudomonas aeruginosa, with oxyclozanide also susceptible to efflux. Synergy was apparent for the combinations against multidrug-resistant clinical isolates of P. aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae. Susceptibility breakpoints for colistin, but also with polymyxin B, were reached upon addition of 1 µg ml-1 of the corresponding salicylanilide against colistin-resistant Gram-negative bacilli. Furthermore, enhanced bacterial killing was observed in all combinations. Our data corroborate the repositioning of the three salicylanilides as adjuvants to counter resistance to the antibiotic of last resort colistin. Our findings are timely and relevant since the global dissemination of plasmid-mediated colistin resistance had been realized.

The Anthelmintic Drug Niclosamide Synergizes with Colistin and Reverses Colistin Resistance in Gram-Negative Bacilli.

There is an urgent need for new therapies to overcome antimicrobial resistance especially in Gram-negative bacilli (GNB). Repurposing old U.S. Food and Drug Administration-approved drugs as complementary agents to existing antibiotics in a synergistic combination presents an attractive strategy. Here, we demonstrate that the anthelmintic drug niclosamide selectively synergized with the lipopeptide antibiotic colistin against colistin-susceptible but more importantly against colistin-resistant GNB, including clinical isolates that harbor the mcr-1 gene. Breakpoints for colistin susceptibility in resistant Gram-negative bacilli were reached in the presence of 1 µg/ml (3 µM) niclosamide. Reversal of colistin resistance was also observed in combinations of niclosamide and polymyxin B. Enhanced bacterial killing was evident for the combination, in comparison to colistin monotherapy, against resistant Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae Accumulating evidence in the literature, along with our results, strongly suggests the potential for the combination of niclosamide and colistin to treat colistin-resistant Gram-negative bacillary infections. Our finding is significant since colistin is an antibiotic of last resort for multidrug-resistant Gram-negative bacterial infections that are nonresponsive to conventional treatments. With the recent global dissemination of plasmid-encoded colistin resistance, the addition of niclosamide to colistin therapy may hold the key to overcome colistin resistance.

Cefiderocol: A Siderophore Cephalosporin with Activity Against Carbapenem-Resistant and Multidrug-Resistant Gram-Negative Bacilli.

Cefiderocol is an injectable siderophore cephalosporin discovered and being developed by Shionogi & Co., Ltd., Japan. As with other ß-lactam antibiotics, the principal antibacterial/bactericidal activity of cefiderocol occurs by inhibition of Gram-negative bacterial cell wall synthesis by binding to penicillin binding proteins; however, it is unique in that it enters the bacterial periplasmic space as a result of its siderophore-like property and has enhanced stability to ß-lactamases. The chemical structure of cefiderocol is similar to both ceftazidime and cefepime, which are third- and fourth-generation cephalosporins, respectively, but with high stability to a variety of ß-lactamases, including AmpC and extended-spectrum ß-lactamases (ESBLs). Cefiderocol has a pyrrolidinium group in the side chain at position 3 like cefepime and a carboxypropanoxyimino group in the side chain at position 7 of the cephem nucleus like ceftazidime. The major difference in the chemical structures of cefiderocol, ceftazidime and cefepime is the presence of a catechol group on the side chain at position 3. Together with the high stability to ß-lactamases, including ESBLs, AmpC and carbapenemases, the microbiological activity of cefiderocol against aerobic Gram-negative bacilli is equal to or superior to that of ceftazidime-avibactam and meropenem, and it is active against a variety of Ambler class A, B, C and D ß-lactamases. Cefiderocol is also more potent than both ceftazidime-avibactam and meropenem versus Acinetobacter baumannii, including meropenem non-susceptible and multidrug-resistant (MDR) isolates. Cefiderocol's activity against meropenem-non-susceptible and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriales is comparable or superior to ceftazidime-avibactam. Cefiderocol is also more potent than both ceftazidime-avibactam and meropenem against all resistance phenotypes of Pseudomonas aeruginosa and against Stenotrophomonas maltophilia. The current dosing regimen being used in phase III studies is 2 g administered intravenously every 8 h (q8 h) using a 3-h infusion. The pharmacokinetics of cefiderocol are best described by a three-compartment linear model. The mean plasma half-life (t½) was ~ 2.3 h, protein binding is 58%, and total drug clearance ranged from 4.6-6.0 L/h for both single- and multi-dose infusions and was primarily renally excreted unchanged (61-71%). Cefiderocol is primarily renally excreted unchanged and clearance correlates with creatinine clearance. Dosage adjustment is thus required for both augmented renal clearance and in patients with moderate to severe renal impairment. In vitro and in vivo pharmacodynamic studies have reported that as with other cephalosporins the pharmacodynamic index that best predicts clinical outcome is the percentage of time that free drug concentrations exceed the minimum inhibitory concentration (%fT > MIC). In vivo efficacy of cefiderocol has been studied in a variety of humanized drug exposure murine and rat models of infection utilizing a variety of MDR and extremely drug resistant strains. Cefiderocol has performed similarly to or has been superior to comparator agents, including ceftazidime and cefepime. A phase II prospective, multicenter, double-blind, randomized clinical trial assessed the safety and efficacy of cefiderocol 2000 mg q8 h versus imipenem/cilastatin 1000 mg q8 h, both administered intravenously for 7-14 days over 1 h, in the treatment of complicated urinary tract infection (cUTI, including pyelonephritis) or acute uncomplicated pyelonephritis in hospitalized adults. A total of 452 patients were initially enrolled in the study, with 303 in the cefiderocol arm and 149 in the imipenem/cilastatin arm. The primary outcome measure was a composite of clinical cure and microbiological eradication at the test-of-cure (TOC) visit, that is, 7 days after the end of treatment in the microbiological intent-to-treat (MITT) population. Secondary outcome measures included microbiological response per pathogen and per patient at early assessment (EA), end of treatment (EOT), TOC, and follow-up (FUP); clinical response per pathogen and per patient at EA, EOT, TOC, and FUP; plasma, urine and concentrations of cefiderocol; and the number of participants with adverse events. The composite of clinical and microbiological response rates was 72.6% (183/252) for cefiderocol and 54.6% (65/119) for imipenem/cilastatin in the MITT population. Clinical response rates per patient at the TOC visit were 89.7% (226/252) for cefiderocol and 87.4% (104/119) for imipenem/cilastatin in the MITT population. Microbiological eradication rates were 73.0% (184/252) for cefiderocol and 56.3% (67/119) for imipenem/cilastatin in the MITT population. Additionally, two phase III clinical trials are currently being conducted by Shionogi & Co., Ltd., Japan. The two trials are evaluating the efficacy of cefiderocol in the treatment of serious infections in adult patients caused by carbapenem-resistant Gram-negative pathogens and evaluating the efficacy of cefiderocol in the treatment of adults with hospital-acquired bacterial pneumonia, ventilator-associated pneumonia or healthcare-associated pneumonia caused by Gram-negative pathogens. Cefiderocol appears to be well tolerated (minor reported adverse effects were gastrointestinal and phlebitis related), with a side effect profile that is comparable to other cephalosporin antimicrobials. Cefiderocol appears to be well positioned to help address the increasing number of infections caused by carbapenem-resistant and MDR Gram-negative bacilli, including ESBL- and carbapenemase-producing strains (including metallo-ß-lactamase producers). A distinguishing feature of cefiderocol is its activity against resistant P. aeruginosa, A. baumannii, S. maltophilia and Burkholderia cepacia.

Dilipid ultrashort cationic lipopeptides as adjuvants for chloramphenicol and other conventional antibiotics against Gram-negative bacteria.

The necessity to develop therapeutic agents and strategies to abate the spread of antibiotic-resistant pathogens is prominent. Antimicrobial peptides (AMPs) provide scaffolds and inspiration for antibiotic development. As an AMP of shorter scaffold, eight dilipid ultrashort cationic lipopeptides (dUSCLs) were prepared consisting of only four amino acids and varying dilipids. Lipids were acylated at the peptide N-terminus and the ε-amine side chain of the N-terminal L-lysine. Compounds that possess aliphatic dilipids of ≥ 11 carbons-long showed significant hemolysis and therefore limited therapeutic application. Several non-hemolytic dUSCLs were identified to enhance the activity of chloramphenicol and other conventional antibiotics against Gram-negative bacteria. Compounds 2 and 6 have a short peptide sequence of KKKK and KKGK, respectively, and are both acylated with an aliphatic dilipid of nine carbons-long potentiated chloramphenicol against MDR clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacteriaceae. Both dUSCLs showed comparable adjuvant potency in combination with chloramphenicol. However, dUSCL 2 synergized with a wider span of antibiotic classes against P. aeruginosa relative to dUSCL 6 that included rifampicin, trimethoprim, minocycline, fosfomycin, piperacillin, ciprofloxacin, levofloxacin, moxifloxacin, linezolid and vancomycin. Our data revealed that dUSCLs can indirectly disrupt active efflux of chloramphenicol in P. aeruginosa. This along with their membrane-permeabilizing properties may explain the dUSCLs synergistic combination with conventional antibiotics against Gram-negative bacteria.

Development of dilipid polymyxins: Investigation on the effect of hydrophobicity through its fatty acyl component.

Continuous development of new antibacterial agents is necessary to counter the problem of antimicrobial resistance. Polymyxins are considered as drugs of last resort to combat multidrug-resistant Gram-negative pathogens. Structural optimization of polymyxins requires an in-depth understanding of its structure and how it relates to its antibacterial activity. Herein, the effect of hydrophobicity was explored by adding a secondary fatty acyl component of varying length onto the polymyxin structure at the amine side-chain of l-diaminobutyric acid at position 1, resulting to the development of dilipid polymyxins. The incorporation of an additional lipid was found to confer polymyxin activity against Gram-positive bacteria, to which polymyxins are inherently inactive against. The dilipid polymyxins showed selective antibacterial activity against Pseudomonas aeruginosa. Moreover, dilipid polymyxin 1 that consists of four carbon-long aliphatic lipids displayed the ability to enhance the antibacterial potency of other antibiotics in combination against P. aeruginosa, resembling the adjuvant activity of the well-known outer membrane permeabilizer polymyxin B nonapeptide (PMBN). Interestingly, our data revealed that dilipid polymyxin 1 and PMBN are substrates for the MexAB-OprM efflux system, and therefore are affected by efflux. In contrast, dilipid polymyxin analogs that consist of longer lipids and colistin were not affected by efflux, suggesting that the lipid component of polymyxin plays an important role in resisting active efflux. Our work described herein provides an understanding to the polymyxin structure that may be used to usher the development of enhanced polymyxin analogs.

Tobramycin-Linked Efflux Pump Inhibitor Conjugates Synergize Fluoroquinolones, Rifampicin and Fosfomycin against Multidrug-Resistant Pseudomonas aeruginosa.

In this study, we examined the in vitro effect of tobramycin-efflux pump inhibitor (TOB-EPI) conjugates in combinations with fluoroquinolones, rifampicin and fosfomycin on the growth of multi-drug resistant (MDR) and extremely-drug resistant (XDR) Pseudomonas aeruginosa. The TOB-EPI conjugates include tobramycin covalently linked to 1-(1-naphthylmethyl)-piperazine (NMP) (1), paroxetine (PAR) (2) and a dibasic peptide analogue of MC-04,124 (DBP) (3). Potent synergism was found for combinations of TOB-NMP (1), TOB-PAR (2) or TOB-DBP (3) with either fluoroquinolones (moxifloxacin, ciprofloxacin), rifampicin or fosfomycin against a panel of multidrug-resistant/extensively drug-resistant (MDR/XDR) P. aeruginosa clinical isolates. In the presence of ≤8 mg/L (6.1⁻7.2 µM) (≤¼ × MICadjuvant) concentration of the three conjugates, the MIC80 of moxifloxacin, ciprofloxacin, rifampicin and fosfomycin were dramatically reduced. Furthermore, the MIC80 of rifampicin (0.25⁻0.5 mg/L) and fosfomycin (8⁻16 mg/L) were reduced below their interpretative susceptibility breakpoints. Our data confirm the ability of TOB-NMP (1), TOB-PAR (2) and TOB-DBP (3) conjugates to strongly synergize with moxifloxacin, ciprofloxacin, rifampicin and fosfomycin against MDR/XDR P. aeruginosa. These synergistic combinations warrant further studies as there is an urgent need to develop new strategies to treat drug-resistant P. aeruginosa infections.

Absolute Quantitation of Glycoforms of Two Human IgG Subclasses Using Synthetic Fc Peptides and Glycopeptides.

Immunoglobulins, such as immunoglobulin G (IgG), are of prime importance in the immune system. Polyclonal human IgG comprises four subclasses, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitative method for these subclasses and their Fc N-glycoforms, (glyco)peptides were synthesized using a solid-phase approach and used as internal standards. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR standards, respectively. For IgG1, a similar glycopeptide where tyrosine (Y) was isotopically labelled was used to quantify monoclonal IgG1 that had been treated with the enzyme Endo-F2, i.e., yielding tryptic glycopeptide EEQYN(GlcNAc)STYR. The next step was to quantify single subclasses within polyclonal human IgG samples. Although ion abundances in the MALDI spectra often showed higher signals for IgG2 than IgG1, depending on the spotting solvent used, determination of amounts using the newly developed quantitative method allowed to obtain accurate concentrations where IgG1 species were predominant. It was observed that simultaneous analysis of IgG1 and IgG2 yielded non-quantitative results and that more success was obtained when subclasses were quantified one by one. More experiments served to assess the respective extraction and ionization efficiencies of EEQYNSTYR/EEQFNSTFR and EEQYN(GlcNAc)STYR/EEQFN(GlcNAc)STFR mixtures under different solvent and concentration conditions. Graphical Abstract ᅟ.

Antibiotic Hybrids: the Next Generation of Agents and Adjuvants against Gram-Negative Pathogens?

The global incidence of drug-resistant Gram-negative bacillary infections has been increasing, and there is a dire need to develop novel strategies to overcome this problem. Intrinsic resistance in Gram-negative bacteria, such as their protective outer membrane and constitutively overexpressed efflux pumps, is a major survival weapon that renders them refractory to current antibiotics. Several potential avenues to overcome this problem have been at the heart of antibiotic drug discovery in the past few decades. We review some of these strategies, with emphasis on antibiotic hybrids either as stand-alone antibacterial agents or as adjuvants that potentiate a primary antibiotic in Gram-negative bacteria. Antibiotic hybrid is defined in this review as a synthetic construct of two or more pharmacophores belonging to an established agent known to elicit a desired antimicrobial effect. The concepts, advances, and challenges of antibiotic hybrids are elaborated in this article. Moreover, we discuss several antibiotic hybrids that were or are in clinical evaluation. Mechanistic insights into how tobramycin-based antibiotic hybrids are able to potentiate legacy antibiotics in multidrug-resistant Gram-negative bacilli are also highlighted. Antibiotic hybrids indeed have a promising future as a therapeutic strategy to overcome drug resistance in Gram-negative pathogens and/or expand the usefulness of our current antibiotic arsenal.

Short Proline-Rich Lipopeptide Potentiates Minocycline and Rifampin against Multidrug- and Extensively Drug-Resistant Pseudomonas aeruginosa.

A series of 16 short proline-rich lipopeptides (SPRLPs) were constructed to mimic longer naturally existing proline-rich antimicrobial peptides. Antibacterial assessment revealed that lipopeptides containing hexadecanoic acid (C16) possess optimal antibacterial activity relative to others with shorter lipid components. SPRLPs were further evaluated for their potential to serve as adjuvants in combination with existing antibiotics to enhance antibacterial activity against drug-resistant Pseudomonas aeruginosa Out of 16 prepared SPRLPs, C12-PRP was found to significantly potentiate the antibiotics minocycline and rifampin against multidrug- and extensively drug-resistant (MDR/XDR) P. aeruginosa clinical isolates. This nonhemolytic C12-PRP is comprised of the heptapeptide sequence PRPRPRP-NH2 acylated to dodecanoic acid (C12) at the N terminus. The adjuvant potency of C12-PRP was apparent by its ability to reduce the MIC of minocycline and rifampin below their interpretative susceptibility breakpoints against MDR/XDR P. aeruginosa An attempt to optimize C12-PRP through peptidomimetic modification was performed by replacing all l- to d-amino acids. C12-PRP demonstrated that it was amenable to optimization, since synergism with minocycline and rifampin were retained. Moreover, C12-PRP displayed no cytotoxicity against human liver carcinoma HepG2 and human embryonic kidney HEK-293 cell lines. Thus, the SPRLP C12-PRP is a lead adjuvant candidate that warrants further optimization. The discovery of agents that are able to resuscitate the activity of existing antibiotics against drug-resistant Gram-negative pathogens, especially P. aeruginosa, is of great clinical interest.

Amphiphilic lysine conjugated to tobramycin synergizes legacy antibiotics against wild-type and multidrug-resistant Pseudomonas aeruginosa.

Peptidomimetic modification is a common route of optimization for biologically active peptides. Previous studies in our group have shown that conjugation of amphiphilic tobramycin to other antibacterials enhance their latent outer membrane permeabilizing and efflux blocking activity toward Gram-negative pathogens including Pseudomonas aeruginosa. Herein, we describe the antimicrobial adjuvant properties of amphiphilic lysine ligated to tobramycin. The most potent amphiphilic lysine-tobramycin conjugate 3 potentiated the antibacterial efficacy of 8 clinically used antibiotics against wild type, multidrug- and extensively drug-resistant P. aeruginosa isolates from Canadian hospitals whereas amphiphilic lysine 4 did not. Antibiotics that are synergistic with conjugate 3 included moxifloxacin, ciprofloxacin, erythromycin, chloramphenicol, trimethoprim, novobiocin, linezolid, and fosfomycin. Out of these 8 antibiotics, novobiocin showed highest synergy.

Polymyxin B3-Tobramycin Hybrids with Pseudomonas aeruginosa-Selective Antibacterial Activity and Strong Potentiation of Rifampicin, Minocycline, and Vancomycin.

There is an urgent need to develop novel antibacterial agents able to eradicate drug-resistant Gram-negative pathogens such as Pseudomonas aeruginosa. Antimicrobial hybrids have emerged as a promising strategy to combat bacterial resistance, as a stand-alone drug but also as an adjuvant in combination with existing antibiotics. Herein, we report for the first time the synthesis and biological evaluation of polymyxin-aminoglycoside heterodimers composed of polymyxin B3 covalently linked to tobramycin via an aliphatic hydrocarbon linker. The polymyxin B3-tobramycin hybrids demonstrate potent activity against carbapenem-resistant as well as multidrug- or extensively drug-resistant (MDR/XDR) P. aeruginosa clinical isolates. Furthermore, the most potent hybrid was able to synergize with currently used antibiotics against wild-type and MDR/XDR P. aeruginosa but also against Acinetobacter baumannii as well. The promising biological activity described herein warrants additional studies into design and development of new antimicrobial hybrids able to surmount the problem of antimicrobial resistance.

A Tobramycin Vector Enhances Synergy and Efficacy of Efflux Pump Inhibitors against Multidrug-Resistant Gram-Negative Bacteria.

Drug efflux mechanisms interact synergistically with the outer membrane permeability barrier of Gram-negative bacteria, leading to intrinsic resistance that presents a major challenge for antibiotic drug development. Efflux pump inhibitors (EPIs) which block the efflux of antibiotics synergize antibiotics, but the clinical development of EPI/antibiotic combination therapy to treat multidrug-resistant (MDR) Gram-negative infections has been challenging. This is in part caused by the inefficiency of current EPIs to penetrate the outer membrane and resist efflux. We demonstrate that conjugation of a tobramycin (TOB) vector to EPIs like NMP, paroxetine, or DBP enhances synergy and efficacy of EPIs in combination with tetracycline antibiotics against MDR Gram-negative bacteria including Pseudomonas aeruginosa. Besides potentiating tetracycline antibiotics, TOB-EPI conjugates can also suppress resistance development to the tetracycline antibiotic minocycline, thereby providing a strategy to develop more effective adjuvants to rescue tetracycline antibiotics from resistance in MDR Gram-negative bacteria.

Amphiphilic Tobramycin-Lysine Conjugates Sensitize Multidrug Resistant Gram-Negative Bacteria to Rifampicin and Minocycline.

Chromosomally encoded low membrane permeability and highly efficient efflux systems are major mechanisms by which Pseudomonas aeruginosa evades antibiotic actions. Our previous reports have shown that amphiphilic tobramycin-fluoroquinolone hybrids can enhance efficacy of fluoroquinolone antibiotics against multidrug-resistant (MDR) P. aeruginosa isolates. Herein, we report on a novel class of tobramycin-lysine conjugates containing an optimized amphiphilic tobramycin-C12 tether that sensitize Gram-negative bacteria to legacy antibiotics. Combination studies indicate the ability of these conjugates to synergize rifampicin and minocycline against MDR and extensively drug resistant (XDR) P. aeruginosa isolates and enhance efficacy of both antibiotics in the Galleria mellonella larvae in vivo infection model. Mode of action studies indicate that the amphiphilic tobramycin-lysine adjuvants enhance outer membrane cell penetration and affect the proton motive force, which energizes efflux pumps. Overall, this study provides a strategy for generating effective antibiotic adjuvants that overcome resistance of rifampicin and minocycline in MDR and XDR Gram-negative bacteria including P. aeruginosa.

Multiplexed azido-group isotopic capture (MAGIC) beads: Selective analysis of azido compounds using a propargyl-based cleavable linker, a proof of concept.

RATIONALE: A cleavable linker is designed and synthesized for the selective capture of azide-containing compounds. This article presents a proof of concept methodology involving the use of peptide-functionalized aminopropyl silica, on which the peptide is constructed by solid-phase peptide synthesis. METHODS: The peptide linker has L-propargylglycine (Pra) at one terminal end to allow the conjugation of azide-containing molecules by copper assisted azide alkyne cycloaddition, also known as click reaction. L-Arginine (Arg) is placed just before Pra to permit the release of the captured product by tryptic cleavage. Three glycine (Gly) residues, as part of the linker, are appended to the silica bead to present a spacer section that allows efficient tryptic cleavage devoid of steric hindrance imposed by the bulky bead. The bead composition is Si-O-propyl-NH-Gly-Gly-Gly-Arg-Pra. RESULTS: This solid-phase material can be used to capture and release azide-functionalized compounds. The beads are first tested on three azido compounds, 2-azido-2-deoxyglucose (ADG), BOC-p-azido-Phe-OH (BAzPhe), where BOC = tert-butoxycarbonyl, and tetraacetylated-N-azidomannosamine (Ac4 ManNAz). Copper-mediated click reaction conditions are used and released products are characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS (MS/MS). CONCLUSIONS: This method allows easy identification of captured compounds based on mass and fragmentation analysis. Moreover, it is useful for the analysis of small azide-containing compounds by MALDI-TOF-MS which may not be possible otherwise due to matrix interferences. The insertion of isotopically labeled Arg residues provides the possibility of multiplex analysis, from which the beads have been called MAGIC (for Multiplexed Azido-Group Isotopic Capture). Copyright © 2016 John Wiley & Sons, Ltd.